Interleukin 2 (IL2) is a protein secreted by lymphocytes and belongs to the class of immune modulating substances called lymphokines. IL2 was first described as T-cell growth factor (TCGF), a lymphokine capable of promoting the proliferation of T lymphocytes (Morgan et al. (1976) Science 193, 1007-1008; Ruscetti et al. (1977) J. Immunol. 119, 131-138). IL2 has been shown to modulate many other immunological effects on lymphoid cells including cytotoxic T-cells, natural killer cells, activated B-cells, and lymphokine activated killer (LAK) cells (Robb (1984) Immunol. Today 5, 203 and references therein). IL2 is used to generate LAK cells that kill fresh tumor cells but not normal cells (Grimm et al. (1982) J. Exp. Med. 155, 1823-1841; Mazumder et al. (1984) J. Exp. Med. 159, 495-507). Recently, treatment of cancer patients by administration of IL2 and autologous LAK cells has demonstrated the potential use of IL2 as an immunotherapeutic agent (Rosenberg et al. (1985) N. Eng. J. Med. 313, 1485-1492).
Molecular cloning of the human IL2 cDNA (Taniguchi et al. (1983) Nature 302, 305-310; Deves et al. (1983) Nucleic Acids Res. 11 4307-4323) has allowed determination of the primary sequence structure of IL2 and the production of large amounts of biologically functional IL2 using heterologous expression systems, including E. coli systems (Ju et al. (1987) J. Biol. Chem. 262 5723-5731 and references therein). Molecular cloning procedures have been used to produce structurally altered forms of IL2 with substitutions and deletions of amino acid residues (Ju et al. op. cit. and references therein). By examining the effects of these structural alterations on the function of IL2, regions of the IL2 protein important for its function have been identified. Biologically functional IL2-derived proteins containing additional amino acid residues at the N-terminus of IL2 have also been reported (Takeda patent EP 1058198; Murphy patent WO 86/0090; Sandoz patent EP 0163603). Previous references show that small deletions or substitutions at the C-terminus of IL2 result in loss of functional activity (Ju et al. op. cit.; Liang et al. (1986) J. Biol. Chem. 261, 334-337).
The present invention provides structurally altered, biologically functional forms of IL2 wherein amino acid residues are substituted within the molecule in such a way as to create N-linked glycosylation sites that are effectively glycosylated when the molecule is expressed in eukaryotic cells.
Recombinant IL2 analogs having potential N-glycosylation sites have been reported in Hofer et al. (1987) Biol. Chem. Hoppe-Seyler 368 (9), 1060. Potential N-glycosylation sites were created by substituting Asn for Thr-3, Lys-97, Glu-100, Asp-109 or Cys-125. In only one case (Asn for Glu-100) was N-glycosylation observed. Amino acid positions 3, 97, 100, 109 and 125 in the numbering system used by Hofer et al. correspond to positions 4, 98, 101, 110 and 126 in the numbering system used in this application.